A human-specific, truncated �7 nicotinic receptor subunitassembles with full-length �7 and forms functional receptorswith different stoichiometriesReceived for publication, December 29, 2017, and in revised form, May 15, 2018 Published, Papers in Press, May 21, 2018, DOI 10.1074/jbc.RA117.001698
Matías Lasala, Jeremías Corradi, Ariana Bruzzone, María del Carmen Esandi, and Cecilia Bouzat1
From the Instituto de Investigaciones Bioquímicas de Bahía Blanca, Departamento de Biología, Bioquímica y Farmacia,Universidad Nacional del Sur-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), 8000 Bahía Blanca, Argentina
Edited by Roger J. Colbran
The cholinergic �7 nicotinic receptor gene, CHRNA7, en-codes a subunit that forms the homopentameric �7 receptor,involved in learning and memory. In humans, exons 5–10 inCHRNA7 are duplicated and fused to the FAM7A genetic ele-ment, giving rise to the hybrid gene CHRFAM7A. Its product,dup�7, is a truncated subunit lacking part of the N-terminalextracellular ligand-binding domain and is associated with neu-rological disorders, including schizophrenia, and immuno-modulation. We combined dup�7 expression on mammaliancells with patch clamp recordings to understand its functionalrole. Transfected cells expressed dup�7 protein, but they exhib-ited neither surface binding of the �7 antagonist �-bungaro-toxin nor responses to acetylcholine (ACh) or to an allostericagonist that binds to the conserved transmembrane region. Todetermine whether dup�7 assembles with �7, we generatedreceptors comprising �7 and dup�7 subunits, one of which wastagged with conductance substitutions that report subunit stoi-chiometry and monitored ACh-elicited channel openings in thepresence of a positive allosteric �7 modulator. We found that �7and dup�7 subunits co-assemble into functional heteromericreceptors, which require at least two �7 subunits for channelopening, and that dup�7’s presence in the pentameric arrange-ment does not affect the duration of the potentiated events com-pared with that of �7. Using an �7 subunit mutant, we foundthat activation of (�7)2(dup�7)3 receptors occurs through AChbinding at the �7/�7 interfacial binding site. Our study contrib-utes to the understanding of the modulation of �7 function bythe human specific, duplicated subunit, associated with humandisorders.
�7 is a homomeric member of the nicotinic receptor(nAChR)2 family, which belongs to the pentameric ligand-
gated ion channel superfamily (1–3). �7 receptors are localizedin the central and peripheral nervous systems as well as in non-neuronal cells. They have pleiotropic effects ranging from themodulation of neurotransmitter release and the induction ofexcitatory impulses in the nervous system to the regulationof inflammatory responses in the immune system (4, 5).Decreased expression and function of �7 has been associatedwith neurological and neurodegenerative disorders, includingAlzheimer’s disease, schizophrenia, bipolar disorder, attentiondeficit hyperactivity disorder, and autism spectrum disorder(6).
Nicotinic receptors contain a large extracellular domain,which carries the agonist-binding site, a transmembraneregion, which is formed by four transmembrane segments ofeach subunit (M1–M4) with the M2 domains forming the wallsof the ion pore, and an intracellular region that contains sitesfor receptor modulation and determinants of channel conduct-ance (3, 5). At the interface between the extracellular and trans-membrane domains, several loops form a network that relaysstructural changes from the binding site toward the pore. Thisregion, named the coupling region, contributes to the funda-mental mechanism of receptor activation (7–9).
The acetylcholine (ACh)-binding sites are located at theinterfaces of the extracellular domains of adjacent subunits.Each binding site is composed of a principal face provided byone subunit, which contributes three loops, named A, B and C,and a complementary face provided by the adjacent subunit,which contributes loops D, E, and F (10, 11). The homomeric �7receptor has five identical ACh-binding sites; however, AChoccupancy of only one site is enough for activation (12).
The �7 receptor subunit gene, CHRNA7, has 10 exons and islocated on the long arm of chromosome 15 (15q13– q14). Ahybrid gene, CHRFAM7A, has arisen from a relatively recentpartial duplication that comprises exons 5 to 10 of the CHRNA7gene and is positioned in the same chromosome, centromericto the CHRNA7 gene by 1.6 Mb, interrupting the genetic ele-ment FAM7A. Interestingly, the CHRFAM7A gene is humanspecific (6, 13, 14). The final protein, dup�7, is a truncatedreceptor subunit that lacks the first 95 amino acid residues ofthe �7 protein, which includes loops D and A of the agonist-binding site, and instead contains 27 amino acid residues from
This work was supported by grants from the Universidad Nacional del Sur(UNS) (to C. B. and M. C. E.), Agencia Nacional de Promoción Científica yTecnológica (ANPCYT) (to C. B. and J. C.), and Consejo Nacional de Investi-gaciones Científicas y Técnicas (CONICET) Argentina (to C. B.). The authorsdeclare that they have no conflicts of interest with the contents of thisarticle.
1 To whom correspondence should be addressed: Instituto de Investigacio-nes Bioquímicas de Bahía Blanca (INIBIBB), CONICET Bahía Blanca, CaminoLa Carrindanga Km 7, 8000 Bahía Blanca, Argentina. E-mail: [email protected].
2 The abbreviations used are: nAChR, nicotinic acetylcholine receptor; �-BTX,�-bungarotoxin; PAM, positive allosteric modulator; ACh, acetylcholine;PNU-120596, N-(5-chloro-2,4-dimethoxyphenyl)-N�-(5-methyl-3-isoxa-
FAM7A at the N-terminal domain (15). The CHRFAM7A geneis located in a complex region on chromosome 15 that includesmany segmental, highly variable, duplications that result in sev-eral different copy number variants. These multiple polymor-phisms are associated with the risk to develop several neurolog-ical and psychiatric disorders, such as schizophrenia, bipolardisorder, autism, and idiopathic epilepsies (16, 17). Among thecopy number variants present in chromosome 15, another var-iant-truncated subunit gene has been described, which shows a2-bp deletion in exon 6 of the CHRFAM7A, which has beenassociated with schizophrenia and P50 sensory gating deficit(13, 16).
High expression of dup�7 also occurs on human leukocytes(17, 18). In primary monocytes and macrophages, lipopolysac-charide treatment down-regulates the expression of dup�7(19). These findings suggest that the duplicated isoform mighthave a role in the immune system and cholinergic anti-inflam-matory pathway. It is also present in a great variety of epithelialcells, confirming a wide distribution of expression of this trun-cated subunit (20).
The dup�7 subunit has been expressed heterologously inmammalian cell lines and Xenopus oocytes in a few studies.Immunological studies in GH4C1 cells suggested that dup�7can reach the cell periphery, although it appeared to be at amore inner location than that of �7 (21). No macroscopicresponses have been detected after exposure to �7 agonists (21,22). The role of dup�7 remains unclear because reduction of �7currents, compatible with a negative modulator role, has beenshown in oocytes (21, 23) but not in neuronal cells (22). Byincorporating fluorescent tags Wang et al. (22) proposed thatdup�7 can assemble with the full-length �7 because both sub-units are membrane localized in close position. The stoichiom-etry of these hybrid receptors and their function remainunknown.
To find a way to identify the possible pentameric �7/dup�7arrangements and determine their functional signature, wecombined cell expression and single-channel recordings withthe electrical fingerprinting strategy (12, 24 –26). Receptorswere generated using combinations of �7 and dup�7 subunits,one of which carries a reporter conductance mutation thatallows defining the subunit stoichiometry of the receptor thatoriginated each single-channel opening event in real time. Ourresults provide novel information about the function of theduplicated �7 subunit, which may have a significant role inimmunomodulation and in the pathophysiology of neurologi-cal disorders.
Results
Heterologous expression of dup�7 on mammalian cells
The cloned dup�7 cDNA includes the 1236-nucleotidesequence previously deposited in NCBI and corresponds to theCHRFAM7A isoform 1 (accession number NM_139320). Theinitiator methionine in exon B of CHRFAM7A will produce aprotein including amino acid residues coded by part of exon B,exon A of the FAM7A gene, and exons 5–10 of CHRNA7 (Fig.1A). The final protein contains the N-terminal domain with 27residues of FAM7A followed by the �7 sequence starting at
amino acid residue 96 and therefore lacks loop A and D of theACh-binding site (Fig. 1A).
To establish that the cloned dup�7 cDNA is translated intothe truncated subunit in our system, BOSC-23 cells were trans-fected with plasmid vectors containing either dup�7 or �7cDNAs together with plasmids encoding the chaperone pro-teins, Ric-3 and NACHO in a 2:1:1 molar ratio, respectively.The expression of �7 and dup�7 subunit proteins was detectedby Western blotting from whole cell lysates using a previouslycharacterized antibody raised against the intracellular M3–M4loop that is common to both subunits (27). Western blottingrevealed bands in the 55–57-kDa range in �7 expressing cellsand in a lower molecular mass range (45–50 kDa) in thosetransfected with dup�7 cDNA. These bands, which were notdetected in nontransfected cells, are compatible with theexpected molecular weights of both subunits (Fig. 1B) (18, 19).Double bands as the ones observed have been described beforefor �7 and might represent forms differing in glycosylation orin other post-translational modifications (28 –30). Thus, ourWestern blotting results confirm that dup�7 is transcribed andtranslated in our heterologous expression system.
dup�7 reduces the number of �-BTX-binding sites whenco-expressed with �7
To detect the presence of surface receptors, transfected cellswere labeled by Alexa Fluor 488/�-BTX, a selective �7 antago-nist, and examined by confocal microscopy. The confocalimages showed high levels of expression of �7 but no binding ofAlexa Fluor 488/�-BTX to cells expressing dup�7 as describedbefore (21) (Fig. 2). Analysis of the membrane fluorescenceintensity revealed reduced fluorescence in cells that co-ex-pressed dup�7 with �7 (�7:dup�7 1:3 cDNA subunit ratio) withrespect to those expressing �7 (Fig. 2).
dup�7 does not mediate functional responses elicited by �7orthosteric or allosteric agonists
To examine function, �7 or dup�7 were expressed inBOSC-23 cells and examined by single-channel recordings. Inthe presence of 100 –500 �M ACh, �7 exhibits single brief open-ings (�0.25 ms) flanked by long closings, or less often, fewopenings in quick succession, known as bursts (Fig. 3A, Table 1)
D A
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Figure 1. dup�7 subunit. A, diagram of �7 and dup�7 subunits, showing theloops that form the ACh-binding site (loops A–F); loops at the coupling region(�1�2, Cys, and �8�9 loops), the transmembrane (TM) region, and the FAM7Apart of dup�7 at the N-terminal domain. B, expression of dup�7 on BOSC-23cells. Lysates of BOSC-23 cells transfected with �7 or dup�7 cDNA wereprobed by Western blotting for the subunit protein. NT, corresponds tolysates from nontransfected cells.
(5, 12, 31). Positive allosteric modulators (PAMs), such as thetype II PAM PNU-120596 (32, 33), are used as tools for increas-ing the probability of channel opening of �7. By slowing theonset of �7 desensitization and trapping activated �7 channelsin an open conformation, PNU-120596 facilitates the detectionof infrequent opening events and, at the same time, allows theaccurate registering of their amplitudes (Fig. 3B) (34, 35). Incells expressing �7, ACh in the presence of PNU-120596 (1 �M)elicited long duration openings separated by brief closings,grouped in bursts, which in turn coalesce into long activationperiods, clusters (�1–3 s) (Table 1, Fig. 3B) (32). The openduration histogram is well-fitted by three exponential compo-nents (Table 1, Fig. 3B). Neither clusters nor isolated openings
were detected in the presence of 100 �M ACh and 1 �M PNU-120596 from cells transfected with dup�7 cDNA together withRic-3 and NACHO (n � 23 recordings from 4 different celltransfections, Fig. 3B), in agreement with the lack of macro-scopic responses previously reported (21, 22). These experi-ments were performed in parallel with single-channel record-ings from cells of the same batch transfected under identicalconditions with �7 cDNA to confirm successful transfectionand receptor expression. As an additional control, we per-formed recordings from cells transfected only with Ric-3 andNACHO cDNAs and showed no channel activity elicited byACh and PNU-120596 (n � 8).
Because dup�7 lacks loops A and D of the agonist-bindingsite we sought to explore activation by an �7 allosteric agonist,4BP-TQS, which binds to the transmembrane region that isconserved between �7 and dup�7 (36). The underlying hypoth-esis is that if the absence of loops A and D of the ACh-bindingsite were the cause of the lack of response, this should be over-
A Mock transfected
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Figure 2. �-BTX labeling of BOSC cells transfected with �7 and/or dup�7.Cells were transfected with �7 cDNA, dup�7 cDNA, or with the 1:3 �7/dup�7cDNA combination. Mock transfected cells correspond to cells transfectedwith irrelevant plasmid DNA. The total amount of DNA per transfection wasnormalized with the addition of irrelevant plasmid DNA. A, representativeconfocal microscopy images showing the membrane fluorescence signalgenerated in transfected cells stained with Alexa Fluor 488-labeled �-BTX. Azoomed image is included at the lower right corner of each panel. Scale barscorrespond to 40 �m for the nonzoomed images and 20 �m for the zoomedimages. B, scatter plot of fluorescence intensity in the region of interest (ROI)of individual transfected cells. Results are expressed as mean � S.D. (n � 15).Different letters (a–c) denote statistically significant differences amonggroups (p � 0.0001; Sidak’s multiple comparisons test).
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Figure 3. Functional responses of �7 or dup�7. BOSC-23 cells were trans-fected with �7 or dup�7 cDNAs together with NACHO and Ric-3 as describedunder “Experimental procedures.” A, representative single-channel currentsactivated by 100 �M ACh from cells transfected with �7. Typical open andburst duration histograms are shown. B, representative traces of ACh-elicitedsingle-channel currents in the presence of 1 �M PNU-120596 from cells trans-fected with �7 (top) or dup�7 cDNAs (bottom). Typical open and cluster dura-tion histograms are shown for �7. C, single channel currents activated by theallosteric agonist, 4BP-TQS (50 �M) from cells transfected with �7 (top) ordup�7 cDNAs (bottom). Typical open and cluster duration histograms areshown for �7. Membrane potential: �70 mV. Filter: 9 kHz for ACh and 3 kHzfor ACh and PNU-120596 or 4BP-TQS. Openings are shown as upward deflec-tions. In all histograms, dashed gray lines correspond to individual compo-nents, and black lines correspond to the sum of components. D, macroscopiccurrents activated by 100 �M ACh or 30 �M 4BP-TQS from cells transfectedwith �7 or dup�7 cDNAs. No currents from dup�7-expressing cells were elic-ited by the allosteric agonist.
come by using a ligand that activates from a conserved site. In�7, 10 –50 �M 4BP-TQS elicits prolonged opening events (65 �24 ms, n � 5) grouped in very long duration clusters (1539 �682 ms, n � 5) (Fig. 3C). Macroscopic currents elicited by thisallosteric agonist decay more slowly than those elicited by ACh(Fig. 3D) (36). Neither single-channel currents (12 patches from3 different cell transfections) nor macroscopic responses (12cells from 3 different transfections) elicited by 4BP-TQS weredetected from cells expressing dup�7 (Fig. 3, C and D). Thus,we conclude that dup�7 cannot form functional receptors.
Functional �7/dup�7 heteromeric receptors
In principle, possible co-assembly of �7 and dup�7 could bedetermined by co-expressing both subunits. However, single-channel activity derived from the homomeric �7 will probablybe the predominant one and functional individual heteromericarrangements will be indistinguishable. What is needed is ameans to unequivocally distinguish functional heteromericreceptors and to directly associate channel openings to hetero-meric �7/dup�7 receptors of a given stoichiometry. Thus, todetermine whether the co-assembly of dup�7 and �7 subunitsleads to functional receptors and to establish the stoichiometryof the functional heteromeric arrangements, we applied theelectrical fingerprinting strategy. The strategy is based on thecombined expression of �7 with an �7 subunit mutant thatcontains three arginine substitutions at the intracellularM3–M4 loop region (�7LC for �7 low conductance, Fig. 4, top).Although �7LC receptors are functional as evidenced bymacroscopic current recordings, single channels cannot bedetected because the amplitude is reduced to undetectable lev-els (12, 24 –26, 37). Because of the brief duration of �7 channels,opening events cannot be fully resolved due to filter bandwidthlimitations. Thus, the strategy needs to be performed in thepresence of a modulator, here PNU-120596, which by increas-ing open channel lifetime allows accurate measurements ofchannel amplitude (35). Recordings of �7 in the presence of 100�M ACh and 1 �M PNU-120596 showed a homogeneous ampli-tude population of 9.8 � 1.7 pA (�70 mV membrane potential;n � 5 patches from 3 different cell transfections) for both indi-vidual opening events and clusters (Fig. 4A, Table 1). Under thesame recording conditions, single channel openings from �7LCreceptors were not detected due to their low amplitude (Fig.4B). When �7LC was expressed together with �7 (1:4 for�7:�7LC cDNA subunit ratio), instead of the homogenousamplitude population of clusters detected for �7 alone, clustersof different amplitudes were observed (Fig. 4C). Clusters can be
grouped into different amplitude classes, which can be well-distinguished from the amplitude histograms (Figs. 4C and 5A).Studies from our and other labs have shown that the differentamplitude populations report the number of low conductancesubunits in each type of pentameric arrangement (12, 26, 37).Thus, amplitude classes of clusters of �4-, 6-, 8-, and 10-pA(�70 mV membrane potential) correspond to receptors con-taining 3, 2, 1, and 0 �7LC subunits, respectively (Fig. 5A) (12).In previous studies we also analyzed a 2-pA class that corre-sponds to receptors containing 4 �7LC subunits (12). Here,information from this class was obtained using the reversecombination (see below).
Clusters in the presence of ACh and 1 �M PNU-120596 werenot detected from cells transfected with �7LC cDNA (Fig. 4B)(n � 19 of 4 different transfections) (12) or with dup�7 cDNA(Fig. 4D, n � 23). In contrast, they were detected from cellsexpressing both subunits (�7LC:dup�7, 1:3, 1:4, and 1:6 cDNAsubunit ratios) (Fig. 4E). This result unequivocally indicatesthat dup�7 assembles with �7LC. It is important to notethat the frequency of the active patches was markedly lowerthan that observed for the �7LC/�7 combination. Under simi-lar transfection conditions, 31% of cell patches showed channelactivity in cells co-transfected with �7LC and dup�7 cDNAs(53 of 171 patches), whereas this percentage increased to about80% in cells transfected with �7LC and �7 cDNA.
The analysis of the amplitudes of clusters obtained fromrecordings of cells expressing the �7LC/dup�7 combinationshowed two predominant classes whose mean amplitude valueswere 4.2 � 0.3 and 5.8 � 0.5 pA (Fig. 5B). In these recordings,we did not analyze the lowest 2-pA amplitude class, whichwould correspond to receptors containing one dup�7 subunitbecause channels of this stoichiometry were better detectedusing the reverse subunit combination (see below). In 27% ofpatches, a few clusters of higher amplitude were detected whoseorigin remains unknown. Because dup�7 conserves the portalamino acid residues responsible for �7 conductance, we caninfer that the relationship between the mean amplitude of eachclass and receptor stoichiometry is for dup�7/�7LC the same asfor �7/�7LC. Thus, we can ensure that the detected 6- and4-pA amplitude classes in the �7LC/dup�7 combination cor-respond to heteromeric receptors containing three and twodup�7 subunits, respectively (Figs. 4E and 5B).
To determine whether heteromeric receptors containingonly one dup�7 subunit are functional and to further confirmthat the three portal amino acid residues in dup�7 govern chan-
Table 1Kinetic properties of �7 and �7/dup�7 receptorsCells expressing the specified subunit combination were used for single-channel recordings. Channels were activated by 100 �M ACh in the absence or presence of 1 �MPNU-120596. For �7, a single �10-pA class is detected. The mean durations of open (�o) and clusters (�cluster) were obtained from the corresponding histograms. Channelevents from (�7LC)2(dup�7)3 and (�7LC)2(�7)3 heteromeric receptors correspond to the 6-pA amplitude class recorded from cells transfected with dup�7 and �7LC or �7and �7LC, respectively (Fig. 4E). The differences of durations among all receptors were not statistically significant (p � 0.23 for �o and 0.62 for �cluster, one-way analysis ofvariance).
Subunit combination Receptor PAM (1 �M) Amplitude class (pA) �0 (ms) �cluster (ms) n
nel amplitude, we introduced the triple mutation (RRR) indup�7 to generate a low conductance dup�7 subunit(dup�7LC). We next transfected BOSC-23 cells with the�7/dup�7LC combination (1:8 cDNA subunit ratio) andrecorded single-channel currents in the presence of 100 �M
ACh and 1 �M PNU-120596. The amplitude of the majority ofclusters in all active patches was �10 pA, which corresponds tothat of homomeric �7 receptors, indicating the prevalence ofthis receptor over heteromers. However, clusters of lower
amplitude, which were not detected in cells transfected with �7alone, were detected. The analysis showed clusters of 10-, 8-,and 6-pA amplitude classes (Fig. 4F), which correspond toreceptors containing zero, one, and two dup�7 subunits,respectively. In these experiments, we did not analyze ampli-tude classes lower than 6 pA, because the corresponding pop-ulations were well-detected with the reverse combination(�7LC/dup�7) (Fig. 4E). Thus, the application of the electricalfingerprinting strategy revealed that dup�7 can assemble with�7 forming functional heteromeric receptors containing one,two, or three dup�7 subunits.
Arrangement of (�7)2(dup�7)3 receptors
The ACh-binding site is located at subunit interfaces (Fig.6A). A conserved tyrosine (Tyr-93) located in loop A of theprincipal face of the binding site has been shown to be essentialfor channel activation (38). Because dup�7 lacks Tyr-93 (seebelow), it is likely that it cannot contribute to an activable prin-cipal face. We explored if it contributes to the complementaryface of the binding site although it lacks loop D. Two possiblepentameric arrangements containing three dup�7 and two �7subunits may be formed, depending on whether the two �7
Figure 4. Electrical fingerprinting for �7/�7LC and dup�7/�7LC hetero-meric receptors. Top, models showing the different amino acid residues atthe intracellular region for �7 and �7LC. Left panels, representative single-channel currents activated by 100 �M ACh � 1 �M PNU-120596 from cellsexpressing �7 (A), �7LC (B), �7 � �7LC (C), dup�7 (D), dup�7 � �7LC (E), anddup�7LC � �7 (F). The traces for the mixed subunit conditions are excerptsfrom the same recording. Membrane potential: �70 mV. Filter: 3 kHz. Channelopenings are shown as upward deflections. The dashed lines indicate theamplitude of the different amplitude classes. Right panels: typical amplitudehistograms for a whole recording constructed with events longer than 1 msare shown with the fitted components.
Figure 5. Amplitude classes for the �7/�7LC and dup�7/�7LC combina-tions. A and B, analysis of the amplitude classes for cells transfected with�7LC and �7 (A) or dup�7 (B) cDNAs. Left: A, plot of mean current amplitudeagainst the number of �7LC subunits in the pentameric arrangement for the�7/�7LC combination. The fitted slope by least-squares method is 1.81 �0.06 pA/LC subunit. Data are plotted as mean � S.D. of n � 7 for amplitudeclass of 10, n � 10 for amplitude classes of 8 and 6 pA, and n � 8 for theamplitude class of 4 pA. B, plot of mean current amplitude against the num-ber of �7LC subunits in the pentameric arrangement for the �7LC/dup�7combination. The fitted slope by the least-squares method is 1.63 � 0.3 pA/LCsubunit. Data are plotted as mean � S.D. of n � 15 for amplitude class of 6 andn � 3 for the amplitude class of 4 pA. Right, representative dot plots showingthe distribution of clusters as a function of their mean amplitude. Recordingswere obtained from cells transfected with �7/�7LC (A) or dup�7/�7LC (B).Each plot corresponds to a single recording, and each point, to a single clus-ter. The number of amplitude classes was determined by the X-means algo-rithm included in the QuB software.
subunits are adjacent or not (Fig. 6B). If the two �7 subunitswere not consecutive, activation would occur through the�7/dup�7 interface where dup�7 should provide an activablecomplementary binding-site face. To test this hypothesis, weco-expressed dup�7 with an �7 subunit carrying a mutationat loop D of the complementary face of the binding site(�7W55T). We have previously shown that ACh does not eliciteither single-channel nor macroscopic currents from cellsexpressing �7W55T receptors (Fig. 6C) (12, 24). We did notdetect any channel activity elicited by 100 �M ACh in the pres-ence of 1 �M PNU-120596 from cells transfected with the�7W55T/dup�7 combination (subunit ratios 1:3 and 3:1, n �10 patches from three different cell transfections) (Fig. 6C).These results indicate that the complementary face of the bind-ing site has to be provided also by the �7 subunit to allow acti-vation. Thus, activation of the �7/dup�7 heteromeric receptoroccurs through the �7/�7 interfacial-binding site. In conse-quence, in the pentameric (�7)2(dup�7)3 arrangement, the two�7 subunits are located consecutively.
Finally, we explored if channel kinetics elicited by 100 �M
ACh in the presence of 1 �M PNU-120596 of (�7)2(dup�7)3
receptors are different to those of �7. To this end, we analyzedthe 6-pA amplitude class of channels recorded from cellsexpressing �7LC and dup�7, which corresponds to receptorscontaining three dup�7 subunits, (�7LC)2(dup�7)3 (Figs. 4Eand 5B). We found that the mean duration of the slowest opencomponent and the mean cluster duration were not statisticallysignificantly different from those of �7 or (�7LC)2(�7)3
obtained from the 6-pA population of the �7LC/�7 combina-tion (Table 1). This analysis indicates that the truncatedsubunit does not alter the channel kinetics of potentiatedreceptors.
Discussion
The expression and function of human �7 can be regulated atdifferent stages and by different mechanisms, such as gene reg-ulation through transcriptional mechanisms (6), co-expressionof chaperone proteins (39, 40), receptor up-regulation (41),interaction with intracellular proteins (42), and allosteric mod-ulation by endogenous compounds (43). Another mechanismof potential modulation involves the partial duplication of theparent gene, an event that is evolutionary new and human spe-cific (6, 44). The mechanism underlying such modulation andthe physiological role of the truncated �7 subunit remainunknown.
To explore if the truncated subunit resulting from geneduplication, dup�7, modulates �7 function, we generated adup�7 cDNA, expressed it on mammalian cells, and decipheredreceptor function by single-channel recordings. By using anovel electrophysiological strategy, our results revealed that: (i)dup�7 alone does not form functional ion channels; (ii) dup�7subunit can assemble with �7 forming a variety of heteromeric�7/dup�7 receptors; (iii) for functional heteromeric �7/dup�7receptors, at least two �7 subunits are required; (iv) activationof heteromeric receptors requires an �7/�7 interfacial-bindingsite; and (v) the kinetic signature of potentiated �7 receptors isnot affected by dup�7.
Western blotting using an antibody against the �7 intracel-lular loop, which is conserved between �7 and dup�7, showedthat dup�7 cDNA is well-translated in BOSC-23 cells. No�-BTX binding was detected in cells transfected with dup�7cDNA, in agreement with previous results obtained in oocytes(21). Although no �-BTX binding was observed, heterologousexpression of dup�7 homomers in the rat cell line GH4C1 aswell as in oocytes was detected using an �7 antibody (21). How-ever, it was described that such expression appeared to be at amore inner location than that of �7, probably within the endo-plasmic reticulum (21). Although the probable absence of asignal peptide of the truncated protein suggests a subcellularlocalization, whether dup�7 homomers are present at the sur-face remains undefined. It is here important to note that a spe-cific dup�7 antibody, which would facilitate its detection, is stillnot available.
It has been previously shown that the presence of dup�7reduced significantly the number of �7 receptors in oocytes(21) but not in neuronal cells (22). However, in the latter systempoor dup�7 translation was reported. In our system, overex-pression of dup�7 (3-fold higher cDNA amount than �7cDNA) reduced �-BTX binding at the membrane level. How-ever, we acknowledge that the level of reduction of fluorescencemediated by the presence of dup�7 may not be accuratelydetermined due to possible bias introduced during the selectionof fluorescent cells. In addition, only cells showing membranefluorescence were analyzed. Assuming that translation andassembly are similar between �7 and dup�7 subunits, the bino-mial distribution indicates that 23% of the receptors should bedup�7 homomers and 39.5% should contain only one �7 sub-unit in cells transfected with 1:3 �7:dup�7 cDNA subunit ratio.Under this scenario, an important reduction in �-BTX bindingshould occur because more than 60% of the receptors would not
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Figure 6. Requirements for (�7)2(dup�7)3 channel activation. A, diagramshowing the �7-binding site for ACh. Mutations were introduced at thecomplementary face (W55T). B, possible subunit arrangements of recep-tors containing three dup�7 and two �7 subunits. The arrow shows thefunctional �7/�7 interfacial-binding site. C, representative single-channelrecordings in the presence of 100 �M ACh � 1 �M PNU-120596 showingthe lack of channel activity in cells transfected with dup�7 and �7W55TcDNA. The black arrows show possible ACh-binding sites in which thecomplementary face is provided by dup�7. Membrane potential: �70 mV.Filter: 3 kHz.
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bind �-BTX and the rest would contain a reduced number ofbinding sites.
It is important to note that although we used Ric-3 andNACHO as chaperones, their actions on dup�7 as well as themost appropriate �7 subunit:chaperone ratio remain unknown.Also, the expression of dup�7 appears to depend on the celltype, i.e. in neurons the dup�7:�7 protein ratio is opposite tothat in immune cells, dup�7 being the major product in thelatter cells (17). Thus, this negative modulation of dup�7 on �7expression observed in the heterologous system might not bestraightforward extrapolated to native systems.
Cells expressing only dup�7 did not show any detectable sin-gle-channel activity elicited by ACh in the presence of thepotent PAM PNU-120596. This result supports the consensusthat dup�7 does not form functional receptors in oocytes andmammalian cells (21, 22). A hypothesis in support of theabsence of response is the lack of an intact ACh-binding site. Toovercome the lack of an intact orthosteric agonist-binding site,we used the �7 allosteric ligand, 4BP-TQS, which binds to thetransmembrane region that is conserved between �7 anddup�7 (36). This ligand mediated strong responses in �7 butdid not elicit neither macroscopic nor single-channel currentsin cells expressing dup�7. These results confirm the absence offunctional dup�7 receptors, which could arise from either theabsence of dup�7 homomeric receptors in the membrane orfrom their inability to function as an ion channel.
To gain further insights into why the truncated subunitcannot form functional channels, we modeled dup�7 usingI-TASSER server (45) (Fig. 7). Interestingly, the FAM7A pep-tide superimposed with loop A in the �7 structural model.However, in dup�7 this region lacks Tyr-93, which is requiredfor �7 activation (38). Also, the model shows the lack of loop Dat the complementary face, which carries Trp-55 that is impor-tant for �7 function (25). Moreover, the lack of functionaldup�7 channels is expected because this subunit also lacks the
�1�2 loop (Fig. 7), which is located at the coupling region and isrequired for channel opening (7–9).
The electrical fingerprinting strategy has been extensivelyused for determining functional stoichiometry of �7-contain-ing receptors (12, 26, 35, 37). This strategy requires the accuratemeasurement of channel amplitude, which acts as the reporterof the stoichiometry of each receptor that originated a singleopening event or cluster. Given the brief open-channel lifetimeof �7, the strategy needs to be performed in the presence of aPAM that by increasing the open duration allows full channelamplitude resolution (12, 35). We chose PNU-120596 becauseit binds to a site that is conserved between �7 and dup�7 and atthe same time it greatly increases opening probability thus facil-itating functional detection of low-expressing receptors (46).
The �7LC carries a triple mutation in determinants of chan-nel conductance, which are located at the loop between M3 andM4 transmembrane segments at the intracellular end of the ionchannel (7, 47, 48). The triple mutation does not affect single-channel kinetics and only decreases �7 channel amplitude toundetectable levels (12, 24). As described in previous studies(12, 26, 37), when �7LC was co-expressed with �7, multiple anddiscrete amplitude classes were detected, each one correspond-ing to a different population of receptors with a fixed number oflow conductance subunits. When instead of �7, dup�7 wasexpressed with �7LC, clusters of different amplitudes activatedby ACh in the presence of PNU-120596 were detected. Thisresult unequivocally indicates the presence of surface �7LC/dup�7 functional receptors because no channel activity wasdetected with either of the two individual subunits. The appli-cation of the electrical fingerprinting strategy revealed thatdup�7 can assemble with �7 forming functional heteromericreceptors composed of one, two, or three dup�7 subunits.
Because loop A with its key tyrosine (Tyr-93) is missing indup�7 we infer that in the �7/dup�7 heteromers the �7 subunitshould provide the principal face of the binding site. The lack
Figure 7. Superimposed molecular models of dup�7 and �7. A, structural alignment of extracellular domains of two adjacent �7/�7 and dup�7/dup�7subunits. Human �7 structural model was created by homology modeling based on the structure of the �7-AChBP chimera (PDB code 5AFM) and the 3D modelof dup�7 was generated by the I-TASSER server (see “Experimental procedures”). The �7 subunits are shown in gray. In dup�7 subunits the region correspond-ing to FAM7A is shown in red and that corresponding to �7 in blue. Binding and interface loops present in both molecules are indicated with blue letters andthose present in �7 but absent in dup�7 with black letters. B, alignment of the �7 and dup�7 sequences (accessions numbers CAD88995 and NP_647536). The�7 sequence does not include the signal peptide. The sequences are identical after amino acid residue 95 of �7. dup�7 sequence corresponding to the FAM7Aregion is in red. Residues for the six binding loops (A–F) and loops at the coupling region are indicated with black and gray lines, respectively. Aromatic residuesreported as essential for �7 agonist response are in gray boxes.
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of functional responses from cells expressing dup�7 and�7W55T, which does not contain a functional complementaryface of the binding site, indicates that this face must also beprovided by �7. Thus, in (�7)2(dup�7)3 receptors, the two �7subunits are located consecutively and activation takes placethrough agonist binding at their interface. The fact that(�7)2(dup�7)3 can be activated despite carrying only one intactagonist-binding site is in close agreement with our previousresults showing that only one functional ACh-binding site issufficient for �7 activation and that the four additional sitesincrease ACh sensitivity (12). It also agrees with reports of �7�2receptors showing that activation of this heteromeric receptortakes place through the �7/�7 interface (49, 50). Thus, itappears that in �7-heteromeric receptors at least one �7/�7interfacial-binding site is required for function.
We also showed that the mean durations of channel openingsand clusters of (�7)2(dup�7)3 elicited by ACh in the presence ofPNU-120596 are identical to those of �7, indicating that, atleast in PNU-120596-potentiated receptors, the kinetics are notaffected by dup�7. Unfortunately, this strategy cannot be per-formed in the absence of a potentiator due to the lack of fullamplitude resolution and the low frequency of opening events(35).
Overall, our electrophysiological results predict that dup�7will functionally operate as a negative modulator of �7 activity.Heteromers containing four dup�7 subunits are nonfunctionaland those with three or less dup�7 subunits, despite being func-tional, have reduced ACh sensitivity due to the reduced numberof active ACh-binding sites (12, 25). An additional negativemodulatory action of dup�7 might be associated with thedecrease of the number of surface �7 receptors (21). However,this may differ between native and heterologous systems due todifferences in gene expression, cell-surface translocation, chan-nel assembly, or chaperones. Thus, our findings encourage toexplore the assembly of heteromers in different human tissues.
Our study has been focused on ionotropic responses. How-ever, �7 has been shown to act as a dual ionotropic/metabo-tropic receptor (5, 42). Considering that the �7 channel-inde-pendent signal transduction is important in anti-inflammatoryresponses and that immune cells show high expression ofdup�7 (17), it will be interesting to determine whetherthe metabotropic activity differs between homomeric and�7/dup�7 heteromeric receptors. From a molecular point ofview, our findings provide novel information regarding �7unique activation, and from a physiological point of view, theyhelp to reveal the still unknown impact of the human-specifictruncated subunit on �7 function.
Experimental procedures
Cloning of dup�7 cDNA
The full-length CHRFAM7A (variant 1: NM_139320.1)cDNA that encodes a 27-amino acid terminus correspondingto the FAM7A sequence (NH2-MQKYCIYQHFQFQLLIQHL-WIAANCDI) and thereafter �7 sequence starting atADERFDA, which corresponds to the end of loop A of the bind-ing site, was synthesized de novo (Biomatik, USA) with appro-priate flanking restriction sites, XbaI and HindIII, for subclon-
ing into the pUC19 plasmid to generate pUC19-dup�7. Thedup�7 cDNA was excised from pUC19 – dup�7 and subclonedinto the cytomegalovirus-based expression vector pRBG4 (51).After cloning, the sequence of dup�7 cloned in pRBG4 wasconfirmed by DNA sequencing using capillary electrophoresis(Instituto de Biotecnología CICCVyA, INTA, Argentina).
Site-directed mutagenesis
Mutations were generated using the QuikChange� Site-di-rected Mutagenesis kit (Agilent, UK). The low conductanceform of human �7 (�7LC) or dup�7 (dup�7LC) containedthree mutations at the intracellular loop (Q428R, E432R,S436R) (12).
Cell expression
BOSC-23 cells, derived from HEK-293 cells (51), were trans-fected by the calcium phosphate procedure with dup�7 and/orhuman �7 cDNA (also subcloned in pRBG4 vector), essentiallyas described previously (52, 53). For the �7LC/dup�7 and�7/dup�7LC combinations the cDNA subunit molar ratiosranged between 1:3 and 1:10 to ensure the incorporation ofdup�7 into the heteromeric receptor. Although the cDNA sub-unit ratio is not directly proportional to the subunit stoichiom-etry of the final receptor, an excess of one subunit over the otheris expected to compensate for less efficient translation orassembly and to bias the receptor population toward pentamerswith an excess of the surplus subunit (24, 54). Plasmids harbor-ing cDNAs of the �7 chaperone proteins Ric-3 and NACHOwere incorporated in all transfections (39, 55).
Confocal fluorescence microscopy
Cells were plated on 12-mm glass coverslips in 35-mmdishes. They were transfected with �7 cDNA (0.3 �g), dup�7cDNA (0.9 �g), or with the combination (1:3) of �7 (0.3 �g) anddup�7 (0.9 �g) cDNAs. Ric-3 (1 �g) and NACHO (1 �g)cDNAs were included during the transfection in all conditions.Mock transfected cells correspond to cells transfected withirrelevant plasmid DNA (1.2 �g). The total amount of DNA pertransfection was normalized with the addition of irrelevantplasmid DNA. After 72 h, cell-surface receptor labeling wascarried out by incubating with �-BTX Alexa Fluor 488 conju-gate (Molecular Probes) at a final concentration of 1 �g/ml for1 h in chilled Dulbecco’s modified Eagle’s medium. Cells werethen fixed with 4% paraformaldehyde. Cultures were then ana-lyzed by phase and fluorescence microscopy, using a NikonEclipse E600 microscope and by laser scanning confocalmicroscopy (LSCM; Leica DMIRE2) with a 20 objective. Flu-orescence intensity, expressed as arbitrary units, was measuredafter manually outlining regions of interest with the softwareImageJ (National Institutes of Health, Bethesda, MD). Themaximum fluorescence intensity of a given region of interestwas measured within the �-BTX-positive region of the cell sur-face, and the maximum fluorescence intensity of an area of thesame size positioned over a �-BTX-negative region outside thecell was subtracted. The average fluorescence intensity valueswere calculated for randomly chosen cells for each experimen-tal condition.
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Western blotting
The �7 antibody used (27) was generously provided by Dr.Cecilia Gotti (CNR Neuroscience Institute, Milan, Italy).
Transfected cells were harvested with PBS and lysed in RIPAbuffer (10 mM Tris, pH 7.5, 150 mM NaCl, 2 mM sodiumorthovanadate, 0,1% SDS, 1% Igepal, 1% sodium deoxycholate)in the presence of proteases inhibitors (phenylmethylsulfonylfluoride and protease inhibitor mixture). Equal amounts of pro-teins (30 �g) were separated on SDS-PAGE and transferred tonitrocellulose. The blots were blocked overnight in 5% nonfatmilk and 0.3% Tween 20 in TBS solution at 4 °C and incubatedfor 2 h with the �7 primary antibody (1–2.5 �g/ml) in 2.5%nonfat milk and 0.15% Tween 20 TBS solution. Immunocom-plexes were revealed by chemiluminescence using 1:1000 dilu-tion of horseradish peroxidase-conjugated appropriate second-ary antibody (Amersham Biosciences, GE Healthcare, LittleChalfont, England). Chemiluminescence detection was per-formed using an enhanced detection solution (1.25 mM lumi-nol, 0,2 mM p-coumaric acid, 0.06% (v/v) hydrogen peroxide,100 mM Tris-HCl, pH 8.8). Immunoblots were exposed to auto-radiographic film (Thermo Scientific, Waltham, MA).
Single-channel recordings
Single-channel recordings were obtained in the cell-attachedpatch configuration (31). The bath and pipette solutions con-tained 142 mM KCl, 5.4 mM NaCl, 1.8 mM CaCl2, 1.7 mM MgCl2,and 10 mM HEPES, pH 7.4. For potentiation, 1 �M PNU-120596was added to the pipette solution with ACh (12).
Single-channel currents were digitized at 5–10-�s intervals,low-pass filtered at a cut-off frequency of 10 kHz using an Axo-patch 200B patch clamp amplifier (Molecular Devices Corp.).Single-channel events were idealized by the half-amplitudethreshold criterion using the program QuB 2.0.0.28 with a dig-ital low-pass filter at 9 kHz. A filter of 3 kHz was used in record-ings with PNU-120596 to facilitate the analysis. The open andclosed time histograms obtained from idealization were fittedby the maximum interval likelihood function in QuB (56, 57),with a dead time of 0.03– 0.1 ms. This analysis was performedon the basis of a kinetic model whose resulting probability den-sity function curves properly fit the histograms following themaximum likelihood criteria. For �7, this analysis was done bysequentially adding an open and/or closed state to a startingC 7 O model to properly fit the corresponding histograms.Final models contained 5– 6 closed states and 3– 4 open statesfor �7 in the presence of ACh plus PNU-120596, or three closedstates and 1–2 open states for �7 in the presence of ACh alone(31, 32, 34).
Clusters were identified as a series of closely separated open-ings preceded and followed by closings longer than a criticalduration. Different critical closed times were calculated bymaximum interval likelihood between each closed compo-nent. Critical times between the third and fourth closedcomponents for �7 in the presence of PNU-120596 (�30 to60 ms) were selected in QuB to chop the idealized data andcreate a subdata set that only contained clusters to definemean cluster duration.
Electrical fingerprinting strategy
To define amplitude classes from receptors generated by co-expression of high and low conductance subunits, all clusterswere selected regardless of their current amplitudes. Amplitudehistograms were then constructed, and the different amplitudeclasses were distinguished. The number of amplitude classesand the mean cluster duration for each class were determinedby the X-means algorithm included in the QuB software.Although up to 4 different amplitude classes were detectedusing the X-means algorithm, not all recordings contain eventsof all classes. Open time histograms were determined asdescribed above for a selected amplitude class.
Results with QuB analysis were similar to those obtainedwith TAC and TAC fit programs (Bruxton Corp., Seattle, WA)as described before (12, 24, 25). Briefly, events were detected bythe half-amplitude threshold criterion using the program TAC.To define amplitude classes, analysis was performed by track-ing events regardless of current amplitude. Amplitude histo-grams were then constructed and fitted by TAC fit. The differ-ent amplitude classes were distinguished by this way inexperiments shown in Fig. 4.
Statistical analysis
Unless otherwise noted, data were presented as mean � S.D.Statistical comparisons were done using pairwise t test or one-way analysis of variance with GraphPad Prism (GraphPad Soft-ware Inc.). Statistically significant differences were establishedat p values � 0.05.
Molecular modeling
A homology model for the extracellular region of human �7receptor was created based on the structure of the �7-AChBPchimera (PDB code 5AFM). The amino acid sequence for thehuman �7 subunit (accession number: CAD88995.1) wasaligned and modeled using MODELLER 9v8 (58). Ten modelswere generated; of these, the one with the lowest energy and thesmallest percentage of amino acid residues in the disallowedregion of the Ramachandran plot was selected. To obtain a 3Dmodel of dup�7 we used the I-TASSER server (45). This serverperforms structure and function prediction for a query aminoacid sequence by a combination of homology modeling, thread-ing, and ab initio modeling. Five models were generated, andthe best model was selected on the base of the C-score. Struc-ture analysis and figures were generated using Discovery StudioVisualizer v4.5 suite (Dassault Systemes BIOVIA, San Diego).
Author contributions—M. L., J. C., M. d. C. E., and C. B. conceptual-ization; M. L., J. C., M. d. C. E., and C. B. formal analysis; M. L., J. C.,M. d. C. E., and C. B. investigation; M. L., J. C., A. B., and C. B. meth-odology; M. L. and J. C. writing-review and editing; M. d. C. E. andC. B. supervision; M. d. C. E. and C. B. writing-original draft; C. B.funding acquisition.
Acknowledgments—NACHO and BOSC-23 cells were generously pro-vided by Dr. Sine (Mayo Clinic) and �7 antibody was generouslyprovided by Dr. Cecilia Gotti. Our special thanks to Dr. LeonardoDionisio for contributing to the early stages of this project.
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